The clinical use of high dose methotrexate in conjunction with citrovorum factor "rescue", especially in treatment of solid tumors, has necessitated a re-evaluation of the pharmacokinetics of methotrexate, particularly with regard to the metabolism of this agent in humans. We have undertaken studies aimed at charcterizing the enzyme in human liver responsible for oxidation of methotrexate to 7-hydroxymethotrexate; our investigations involve development and application of highly sensitive radioisotopic analytical techniques for detection and quantitation of this reaction.